Abstract The critical role of the intestine in total body irradiation injury is an established principle of radiation biology. Acute radiation injury targets rapidly dividing cells prominently those in the bone marrow and intestine; however, it is the intestine, which likely determines the effectiveness of radiation mitigators. The failure of sustained intestinal recovery leads to breakdown of the intestinal barrier, bacterial entry into the bloodstream, infection, sepsis and death. Despite decades of research, the role of intestinal stem cells in the recovery and maintenance of the intestinal barrier function from radiation injury is still not understood. The GI epithelium has the fastest renewal rate in an adult mammal and is driven by the intestinal stem cells (ISCs), whose maintenance and activation is critically regulated by their niche. Our preliminary data indicated that TBI rapidly induces damage in the ISCs, ISC niche, intestinal differentiation and barrier, and our novel CMCR mitigators suppress TBI-induced intestinal damage. Our published work has demonstrated that cell death and cell cycle kinetics plays a key role in intestinal recovery from TBI. The central hypothesis of this project is that successful radiation mitigation suppresses irreparable GI damage and ?radiation disease? by preventing ISC dysfunctions through modulation of cell death and cell cycle kinetics. Project 4 will use novel laboratory tools, including in vivo and in vitro intestinal culture models, molecular and imaging analyses, and system biology, to define the role of intestinal stem cell-intrinsic and niche-based mechanisms of these novel mitigators. SA1. Establish intestinal stem cells as a key target of radiation mitigation. SA2. Define the mechanisms of radiation mitigation by preventing ISC dysfunctions. SA3. Investigate the actions of radiation mitigators in isolated ISCs and 3D intestinal culture. We believe that successful completion of these aims will provide novel mechanistic insights to the pathophysiology of the ?radiation disease?. This will help us understand the basis of mitigating agents already developed in the CMCR and new agents in Project 2 (Valerian Kagan, Ph.D., P.I.) and Project 3 (Hulya Bayir, M.D., P.I.) with single agents, and sequential delivery of different classes of agents in Project 1 (Joel S. Greenberger, M.D., P.I.). This project will discover new radiation mitigators in collaboration with Core C Radiobiological Standardization (Michael Epperly, Ph.D.) and Core B Innovative Medicinal Chemistry (Peter Wipf, Ph.D./Detcho Stoyanovsky, Ph.D., Co-PIs) by specifically targeting ISCs.